Genetic information is preserved through generations by chromosome duplication during S-phase DNA replication, which is highly accurate due to the fidelity of replicative polymerases and efficient elimination of replication errors by polymerase-coupled proofreading activity and post-replicative mismatch repair (MMR). Aside from scheduled DNA replication during S-phase, DNA synthesis is also a part of various types of DNA repair, such as nucleotide-excision repair, base-excision repair, and double-strand break (DSB) repair. It has been shown that short-patch synthesis associated with repair of various kinds of DNA damage is highly error-prone 
, making these events important contributors to a cell's overall mutation rate.
DSBs as a source of hypermutability have been documented for several repair events, including gene conversion (GC) and single-strand annealing in vegetative cells 
, and DSB repair in meiosis and non-dividing cells 
. Also, increased mutability has been associated with senescence in telomerase-deficient cells 
, where shortened chromosome ends behave similarly to DSB ends. At least two mechanisms were demonstrated to contribute to DSB-induced mutagenesis. First, unrepaired lesions accumulated in tracts of single-stranded DNA that form after a DSB result in error-prone restoration of the duplex molecule 
. A similar pathway was shown to be responsible for hypermutagenesis associated with recovery of dysfunctional telomeres 
. Second, it has been demonstrated that copying of a donor sequence associated with GC is mutagenic 
, which could be explained by inefficient MMR during GC 
, or by an unusual, conservative mode of synthesis that proceeds without formation of a replication fork 
This study was designed to determine the mutation rate associated with a unique cellular process, break-induced replication (BIR), which is a processive type of DNA replication that can duplicate large chromosomal regions comparable in size to replicons. In stark contrast to S-phase replication, BIR is initiated at a DSB site rather than at a replication origin. BIR proceeds by invasion of one DSB end into the homologous template, followed by initiation of DNA synthesis that can continue for hundreds of kilobases. A variety of repair processes is believed to proceed via BIR, including repair of collapsed replication forks and stabilization of uncapped telomeres. BIR can also repair DSBs produced such that either only one of the two free DNA ends can find homology for strand invasion or both ends can find homology but only in different areas of the genome (reviewed in 
). Notably, a significant fraction of DSB gap repair events also proceed through BIR 
. The occurrence of BIR often leads to loss of heterozygosity (LOH), chromosomal translocations, and alternative telomere lengthening 
, which are genetic instabilities associated with cancer in humans.
Unlike other forms of DSB repair, BIR is believed to proceed in the context of a replication fork 
, and the establishment of the BIR fork requires almost all of the proteins required for initiation of normal replication 
. However, several observations indicate that the BIR replication fork may differ from an S-phase replication fork in several important ways. For example, it has been shown that, in Saccharomyces cerevisiae
, BIR requires Pol32p, a subunit of polymerase δ (Pol δ; 
) that is dispensable for yeast S-phase DNA replication. Further, the roles of the main replicative polymerases may differ between BIR and S-phase replication. Thus, for BIR initiation, only α-primase and Pol δ are essential, while polymerase ε (Pol ε) is involved only in later steps of BIR, and up to 25% of BIR events can complete in the absence of Pol ε 
. Also, BIR initiation is very slow (takes approximately 4 h 
) and is associated with frequent template switching that subsides after the first 10 kb of synthesis 
, which led to speculation that there may be slow assembly of an unstable replication fork that shifts to a more stable version later in synthesis. Alternatively, initiation of BIR might be slow due to a “recombination execution checkpoint” that regulates the initiation of DNA synthesis during BIR 
. All of these unique features of BIR led us to test whether it is more mutagenic than S-phase replication.
Here we demonstrate that DNA synthesis associated with BIR is highly error-prone, as the frequency of frameshift mutations associated with BIR is dramatically increased compared to normal DNA replication. Our results indicate that BIR mutagenesis results from several problems, including increased polymerase error rate and reduced efficiency of MMR.